Of the polymorphisms represented by a single nucleotide polymorphism (SNP), there is a polymorphism that can be used as a mutation characterizing a variation in a homogenous organism. More specifically, a predetermined variation in a homogenous organism can be distinguished from other variations by detecting and identifying a specific mutation such as a polymorphism in genomic DNA. Furthermore, a variation of an organism to be tested can be specified by detecting and identifying the mutation.
As a method for detecting such a mutation in genomic DNA, a method of directly determining a sequence of a mutation site, a method of using a restriction enzyme fragment length polymorphism (RFLP), a method of using an amplification fragment length polymorphism (AFLP) and the like are known. In addition, a method of analyzing a variation based on identification of a polymorphism using a DNA microarray called as a DArT (Diversity Array Technology) method (Nucleic Acids Research, 2001, Vol. 29, No. 4, e25) is known.
A method for preparing a DNA microarray for use in the DArT method is shown in FIG. 9. First, genomic DNA is extracted from a predetermined organism species and fractionated with restriction enzyme A and restriction enzyme B. Next, to the both ends of each of the genomic DNA fragments obtained by the restriction enzyme treatment, an adaptor is connected and each of the genomic DNA fragments is cloned into a vector. Next, using a primer capable of hybridizing with the adaptor, genomic DNA fragments are amplified by PCR. Then, genomic DNA fragments amplified are spotted on a substrate as a probe to prepare a DNA microarray.
Using the DNA microarray thus prepared, a variation of a organism species to be tested can be analyzed. First, genomic DNA is extracted from an organism to be tested, and fractionated with restriction enzyme A and restriction enzyme B that are used for preparing the DNA microarray. To the genomic DNA fragments, an adaptor is connected similarly in the preparation of the DNA microarray and the resultant fragments are amplified by PCR. The amplified genomic DNA fragments are tagged with a fluorescent label etc. and hybridized with the probe spotted on the DNA microarray. Based on the presence or absence of hybridization of the labeled genomic DNA fragment with the probe detected, a difference between the predetermined organism species used in preparation of the DNA microarray and the organism species to be tested can be analyzed.